Project Summary We will continue to design, construct and characterize genetic circuits. We will use micro fluidic tools to grow and observe single cells and colonies in precisely controlled environmental conditions, and we will test a subset of the engineered bacterial strains as therapies in animal models. Single cell and colony dynamics will inform mathematical models that will be used to identify key design characteristics, which will then be rigorously tested using previously established molecular biology techniques. Eight graduate students and postdocs will work on multiple aspects of the project, while maintaining a particular focus on modeling or technology development for monitoring bacteria or in vivo characterization. Our track record demonstrates our ability to train personnel in a multi-disciplinary approach that has led to new tools for Synthetic Biology, along with an increased understanding of gene and signaling networks generally. Our recent characterization of bacterial circuits in animal models has served to highlight the need for beneficial strains that are stable and safe over therapeutically relevant timescales. Accordingly, our Specific Aims focus on stability (Aim 1), delivery (Aim 2), safety (Aim 3), and in vivo testing (Aim 4). Gene circuits inevitably generate mutations that are selected to decrease the additional burden created by the inserted genetic machinery. Our first aim will develop strategies for extending the lifetime of gene circuits in bacteria before selective pressure disables their desired functionality. We will develop computational models and experimentally quantify how circuit redundancy increases circuit lifetime. We will use our experimental platform to monitor functionality across scales from single-cell to batch culture environments. Our second aim will primarily focus on engineering small bacterial ecologies. Here we will use modeling to guide the design of up to three interacting strains that can deliver therapies in a predetermined sequential order. In the third aim, we will build a safety circuit that triggers the death of all bacteria at a given threshold population density. The goal is to create an irreversible intracellular switch that rapidly and efficiently kills all cells before mutations can compromise the safety strategy. In the final aim, we will test the circuits designed in the first three aims in animal models. We will engineer optical markers that enable characterization of the dynamics of bacterial colonies and tumor size in vivo. Importantly, the relative ease and low cost of bacterial cloning will inevitably lead to a bottleneck for the field of Synthetic Biology, as therapeutic strains can be created at a rate that will far exceed the ability to test them. This highlights an acute need for quantitative models that have been thoroughly validated using in vitro technologies. Consequently, only a fraction of the circuits built in Aims 1-3 will be deemed worthy of in vivo testing. More generally, we anticipate that the computational models arising from these studies will be generally applicable across a wide range of emerging applications that employ bacteria.